Despite great advances in AIDS diagnosis and treatment, the continuing AIDS epidemic demands continuing efforts to understand all aspects of HIV replication and to develop new methods for its inhibition. In pursuit of these goals, we have sought to define the activities and interactions of the HIV-1 structural (Gag) proteins so as to design antivirals that interfere with these functions. The Gag proteins are attractive targets since they perform multiple roles during the life cycle. The proteins initially are synthesized as N-terminally myristoylated precursor (PrGag) proteins that employ their N-terminal matrix (MA) domains to target delivery to plasma membrane (PM) virus assembly sites. Evidence indicates that the HIV-1 MA preferentially binds the signaling phospholipid phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2), and that virus membranes are enriched for lipid raft constituents such as cholesterol, sphingomyelin, and ceremide. MA also binds RNA, suggesting a model in which RNA binding protects MA from binding to inappropriate intracellular membranes prior to PrGag delivery to PI(4,5)P2-rich sites at the PM. In addition to its trafficking role, MA also has been shown to mediate the incorporation of the HIV-1 envelope (Env) glycoprotein complex into virus particles. The MA-Env interaction involves the long cytoplasmic tail (CT) of the transmembrane (TM; gp41) portion of Env, and residues at the distal ends (spokes) and interface regions (hubs) of MA trimers; but models for how MA mediates Env assembly into virions remain a matter of speculation. Using our previous studies and preliminary results as a foundation, we propose to dissect the mechanisms of MA-membrane/RNA and MA-Env binding, and to characterize methods for their inhibition. Our results will help clarify how the HIV assembly machinery operates; and will lead to the development of Gag-targeted antivirals, and an understanding of how they work. To achieve these ends, our aims are as follows: 1. Characterization of HIV-1 matrix-membrane/RNA binding and its inhibition. 2. Elucidation of HIV-1 matrix-envelope protein interactions.